Chemically modified TNF-α

ABSTRACT

The present invention concerns a chemically modified TNF-α, and a pharmaceutical composition and a vaccine composition containing the chemically modified TNF-α which are useful for combating overproduction of native TNF-α.

REFERENCE TO RELATED APPLICATIONS

The present application is the national stage under 35 U.S.C. §371 ofinternational application PCT/FR00/01043, filed Apr. 20, 2000 whichdesignated the United States, and which application was not published inthe English language.

The present invention relates to new inactivated cytokines, which can beused for human immunisation.

PCT/FR92100544 has disclosed that it was possible to immunise managainst cytokines after having inactivated them or inactivated native orhomologous peptide fragments with an appropriate treatment preservingtheir immunogenicity. This document more specifically describes thechemical treatment of the cytokine or peptide or homologous fragmentusing an aldehyde or β propiolactone.

These compounds have proven to be very active. They have in particularbeen used within the scope of clinical trials on patients infected withHIV, and the latter have shown some clinical benefits (see A. Gringeriet al. J of AIDS 20: 358-370-1999).

But these compounds, prepared by treatment with formaldehyde, aresometimes difficult to produce, particularly with regard to stabilityand reproducibility.

The applicant has surprisingly discovered that some chemical treatmentsmade it possible to obtain compounds possessing the same immunogenicity,in particular with a greater stability and reproducibility ofpreparation.

That is why a subject of the present application is a cytokine orcytokine fragment derivative of formulaCy—(X—R)_(n)  (I)in which Cy represents a cytokine or a cytokine fragment, —(X—R)represents a free function of an amino acid constituting the cytokine orthe cytokine fragment where X represents an NH group or a sulphur atom,n represents an integer from 1 to 70, and R represents a chemical grouppreserving for the said cytokine or said fragment sufficient immunogenicproperties to create antibodies neutralising or blocking the said nativecytokine and at the same time bringing about the loss of at least 90% ofthe biological properties of the said native cytokine, with theexception of the product of the reaction of a native cytokine with analdehyde and carboxymethylated interferon-α.

In preferential conditions for the implementation of the above-describedinvention, R is a group which fixes itself to a thiol function and Xrepresents a sulphur atom group.

R is thus preferably a carboxymethyl, carboxamide, N-alkylmaleimide,thionitrobenzoic group or a radical formed by reaction of a thiolfunction with ammonium 4-chloro-7-sulphobenzofurazane,N-[iodoethyl]-trifluoroacetamide r orN-(6-[7-amino-4-methylcoumarin-3-acetamido]hexyl)-3-(2′-pyridyldithio)propionamide.

Carboxymethylation blocks the cysteine residues, by adding acarboxymethyl radical (treatment of cytokine with iodoacetic acid, forexample), carboxyamidination adds a carboxamide radical (treatment ofthe cytokine with iodoacetamide, for example), maleimidation adds anN-alkylmaleimide radical to the cysteines, for example N-ethylmaleimide(treatment of the cytokine with N-ethylmaleimide), Ellman's reactionadds a thionitrobenzoic radical to the cysteine (treatment of thecytokine with 5,5′-dithio-bis-[2-nitrobenzoic] acid or DTNB).

In other preferential conditions for the implementation of theabove-described invention, R is a group which fixes itself to an aminofunction and X represents an NH group.

R is thus preferably a radical formed by reaction of an amino functionwith ethylacetimidate, an anhydride, 2-iminothiolane-HCl, N-succinimidylS-acetylthioacetate, sulphosuccinimidyl-acetate,sulphosuccinimidyl-4-0-[4,4′-dimethoxytrityl]butyrate, succinimidyl7-amino-4-methylcoumarin-3-acetate or sulphosuccinimidyl7-amino-4-methylcoumarin-3-acetate or phenylglyoxal.

R advantageously has a molecular weight of between 10 and 2000,preferably between 20 and 1500, in particular between 30 and 1000,especially between 40 and 500.

R is preferably an organic radical as has been seen above.

The treatment of cytokine with ethylacetimidate produces the amidinationof the amino functions. Citraconic anhydride can be mentioned as auseable anhydride.

As has been seen, other reagents can act on the amine functions such asfor example Traut's reagent (treatment with 2-iminothiolane-HCl), by theaddition of an SH protected by N-succinimidyl S-acetilthioacetate, orsulphosuccinimidyl-acetate orsulphosuccinimidyl-4-0-[4,4′-dimethoxytrityl]butyrate, or alsosuccinimidyl 7-amino-4-methylcoumarin-3-acetate or sulphosuccinimidyl7-amino-4-methylcoumarin-3-acetate.

These chemical inactivations are achieved by a chemical modification ofcertain residues of the cytokine which blocks the biological function ofthe native cytokine. Preferably 50% of the above X groups above aremodified, in particular 70%, especially all of them. The groups where Xrepresents NH and those where X represents a sulphur atom, can bemodified at the same time or preferably only one of these two groups.“n” can range up to 10 for example in the case where X represents asulphur atom and for example up to 70 for example in the case where Xrepresents NH.

The immunogenic compound according to the invention can be constitutedby all or a fragment of the cytokine and can comprise, as is well knownto a person skilled in the art, one or more modifications in the aminoacids of this protein or fragment such as deletions, substitutions,additions, or functionalisations such as acylation of amino acids, in sofar as these modifications remain within the framework specified above(absence of toxicity, immunological characteristics). For example, ingeneral the replacement of a leucine residue by an isoleucine residuedoes not modify such properties. The modifications must generallyconcern less than 30% of the amino acids, preferably less than 20% andquite particularly less than 10%.

A fragment can comprise from 8 to 110 amino acids for example,preferably from 12 to 60 amino acids and in particular from 12 to 40amino acids. Such a fragment can also comprise a C or N terminal side offrom 1 to 5 supplementary amino acids that is to say different from theoriginal segment.

Generally speaking, as far as modifications are concerned, the homologyor the similarity between the modified immunogen and the cytokine a 10or part of the native cytokine, as well as the dimensions of theimmunogenic compound, as well as the methods of use, or coupling of theimmunogenic compound according to the invention to an immunogenicprotein such as tetanic toxoid, reference may be made in particular toWO-A-86/06 414 or EP-A-0.220.273 or PCT/US.86/00831, which areequivalents, the teaching of which is incorporated here by way ofreference.

A subject of the present invention is also a preparation process for acytokine or a fragment as described above, characterised in that thesaid cytokine or the said fragment is subjected to the action of areagent capable of grafting an R group onto the said cytokine or thesaid fragment, in order to obtain the expected compound which isisolated.

As the examples described in the experimental part below and carried outon cytokines of human origin show, the new products obtained by thesetreatments, called “kinoids”, have lost their natural biologicalactivity, and are capable of inducing an immune response in mammals. Inaddition, the antibodies obtained by using these kinoids as immunogensrecognise the non-inactivated native protein.

The examples cited refer to cytokines, IFNγ, TNFα, IL1, IL4, IL6, IL10,IL13 but these reactions can be applied to all the cytokines(interleukins—IL1, IL2, IL3 etc . . . , transforming growthfactors—TGF-α and β, interferons, α, β, γ, τ (tau), tumor necrosisfactors—TNF α and β, chemokines etc . . . ). The cytokine or ahomologous peptide fragment can be produced by chemical synthesis oralso by genetic engineering. By homologous peptide fragment is meant apeptide sequence which is sufficiently similar to the native cytokine toinduce crossed antibodies which will neutralise the biological activityof the native cytokine. For example, the homologous peptide fragment canbe a cytokine mutant prepared by genetic engineering. The advantage ofchemical inactivation is that it will in addition make it possible tostabilise the molecule in order to obtain reproducible conformations andpreparations.

In order to maximise the anti-cytokine immune response, the use ofkinoids inactivated in a different manner and which can be complementaryin terms of the immune reaction that they induce can be combined: forexample a kinoid prepared by a method targeting the cysteines which canbe combined with a kinoid prepared according to a method targeting theamine residues.

The kinoids can be combined to combat overproduction of cytokines, notonly of kinoids of the same cytokine inactivated by different methods,but also kinoids of different cytokines overproduced in the sameillness: for example in the case of AIDS immunisation can be carried outusing combined kinoids IFNα and IFNγ, or in rheumatoid polyarthritisusing kinoids IL1 and TNFα.

The compounds which are the subject of the present invention possessvery beneficial pharmacological properties. They are particularlybeneficial for active anti-cytokine immunisation in the case whereillnesses are combined with an overproduction of these cytokines.

These properties are illustrated below in the experimental part. Theyjustify the use of the compounds of formula (I) described above, as amedicament.

That is why a subject of the invention is also the compoundscorresponding to formula ICy—(X—R)_(n)  (I)in which Cy represents a cytokine or a cytokine fragment, —(X—R)represents a free function of an amino acid constituting the cytokine orthe cytokine fragment where X represents an NH group or a sulphur atom,n represents an integer from 1 to 70, and R represents a chemical group,preserving for the said cytokine derivative or said fragment sufficientimmunogenic properties to create antibodies neutralising or blocking thesaid native cytokine and at the same time bringing about the loss of atleast 90% of the biological properties of the said native cytokine, fortheir use in a method of therapeutic treatment of the human or animalbody, that is to say as a medicament.

The medicaments according to the present invention are used intreatments which are both curative and preventative. Generally speaking,they can be used in pathologies linked to an overproduction, orundesirable production of cytokine. For example, the TNFα kinoid can beused in active immunisation in rheumatoid polyarthritis where it hasalready been shown that the blocking of TNF by antibodies (passiveimmunisation) or soluble receptors limits the development of theillness, or also in cancers when there is overproduction of TNFα andcachexia. The kinoid IL1 can also be used in rheumatoid polyarthritiswhere there is an overproduction of IL1. The kinoid IFNγ can be used inAIDS where there is an overproduction of IFNγ: it can be used inmultiple sclerosis where it has been shown that the administration ofIFNγ leads to a worsening of the illness, or also in juvenile diabeteswhere it has been clearly shown on animal subjects and also by in situstudies in humans that the destruction of the islets of Langerhans isdue to an overproduction of IFNγ. The kinoid IFNα can be used in AIDSwhere its overproduction has been clearly established and where theclinical benefit of anti-IFNα immunisation has been shown or also inlupus erythematous which has been shown to be able to be triggered by aninjection of IFNα. The kinoid IL6 can be used in multiple myeloma orCastleman's disease or some melanomas where it has been shown that thecancerous cells proliferate thanks to IL6. The kinoids IL4 and IL13,isolated or together, can be used in cases of allergic pathologies whereit has been shown that these cytokines are over-produced. All of thesenon-limiting examples show the wide range of uses of the kinoidsdescribed in this patent.

As in any active immunisation, the treatment can use only a fragment ofcytokine inactivated by the methods mentioned above to stimulate theimmune reaction which will neutralise the biological activity of thecytokine.

The immunogenic compounds according to the invention can be used asfollows:

An immunogenic compound according to the present invention isadministered to a patient, for example by subcutaneous or intramuscularroute, in a sufficient quantity to be effective on a therapeutic level,to a subject in need of such a treatment. The dose administered canrange for example from 20 to 1000 μg by intramuscular route in the formof w/o emulsion, once a month over three months, then periodicallyaccording to the level of the serum antibodies induced, for exampleevery 2-6 months.

A composition according to the invention can be administered by anyconventional route in use in the field of vaccines, in particular bysubcutaneous route, by intramuscular route, by intravenous route or byoral route. The oromucous membrane or perriasal route can also be cited.Administration can take place in a single dose or be repeated one ormore times after a certain time period.

A subject of the invention is also pharmaceutical compositions whichcontain at least one aforementioned compound, as an active ingredient.

In these compositions, the active ingredient is advantageously presentin physiologically effective doses; the aforementioned compositionscontain in particular an effective immunogenic dose of at least oneactive ingredient above. The immunogenic compound can be presented onits own or mixed with a pharmaceutically acceptable excipient such as anadjuvant.

These pharmaceutical compositions can in particular be liquids and canbe presented in all the pharmaceutical forms currently used in humanmedicine for vaccines, such as for example injectable preparations inparticular in the form of an emulsion; they are prepared according tothe usual methods. The active ingredient(s) can be incorporated intoexcipients normally used in these pharmaceutical compositions, such asaqueous vehicles, calcium phosphate, alum . . .

A subject of the present invention is also a preparation process for anabove-described composition, characterised in that the activeingredient(s) is mixed, according to methods known per se, withacceptable, in particular pharmaceutically acceptable, excipients.

Finally, a subject of the invention is the use of a cytokine or acytokine fragment of formulaCy—(X—R)_(n)  (I)in which Cy represents a cytokine or a cytokine fragment, —(X—R)represents a free function of an amino acid constituting the cytokine orthe cytokine fragment where X represents an NH group or a sulphur atom,n represents an integer from 1 to 70, and R represents a chemical grouppreserving for the said cytokine or said fragment sufficient immunogenicproperties to create antibodies neutralising or blocking the said nativecytokine and at the same time bringing about the loss of at least 90% ofthe biological properties of the said native cytokine, in order toobtain a medicament intended to combat the overproduction of thesenative cytokines.

In addition a subject of the invention is a kit comprising apharmaceutical vaccine composition which in addition to the activeingredient (for example cytokine or cytokine fragment) can comprise anadjuvant and/or another immunogen with the anti-overproductionproperties in respect of these native cytokines.

Preferential conditions for the use of the cytokines or cytokinefragments described above apply equally to the other subjects of theinvention mentioned above.

The following examples illustrate the present application.

EXAMPLE 1 Treatment of IFNγ by Maleimidation

10 mg of human recombinant IFNγ was placed in solution at 0.5 mg per mlin 0.1 M pH 7 phosphate buffer. 1 mg of N-ethylmaleimide was added tothis solution. After 1 hour of reaction, the solution was dialysedagainst pH 7.2 PBS. The analysis of the kinoid with acrylamide gel showsthe same 17 KD band compared with the native IFNγ.

EXAMPLE 2 Loss of the Biological Activity of the Inactivated IFNγ byMaleimidation

The kinoid IFNγ was then tested to evaluate its biological activity.There are several biological activity tests for IFNγ. Tests forapoptosis induction, tests for induction of surface antigens such as MHC(major histocompatibility complex), but also testing for an antiviraleffect on MDBK cells.

The aim of the antiviral effect measurement test is to evaluate theinhibition of the lysis of MDBK cells in the presence of IFNγ. This testis very sensitive and makes it possible to measure IFNγ picograms.

Freshly trypsinated MDBK cells are grown in RPMI medium, 5% foetal calfserum, at a concentration of 30 000 cells per well. After at least 6hours, the cells are washed in RPMI, then 50 ml of IFNγ is added,diluted in RPMI in a series of ½ dilutions. The plate is then incubatedat 37° C. 5% CO₂ over 18 to 24 hours.

After 18 to 24 hours, the supernatant of each well is removed, the cellsare washed and 100 ml containing 100 LD50 (lethal dose 50%) of VSV virusdiluted in RPMI is added. The plate is left to incubate over 18 to 36hours. The lytic effect of the virus is measured under the microscopeand makes it possible to quantify (scale 1 to 4) from which dilution thelysis operates.

In order to test the activity of the kinoid compared with the nativeIFNγ, solutions containing 100 ng/ml of these molecules are used. Thefollowing result is obtained:

Native IFNγ Maleimidated kinoid IFNγ Positive antiviral effect Up to adilution of Up to a dilution of 2² 2¹⁰

The kinoid has thus lost its antiviral activity and is inactive at afactor greater than 2⁸ in these conditions, which corresponds to aninactivation greater than 99%.

EXAMPLE 3 Immunogenicity of the Maleimidated IFNγ

Kinoids prepared according to example 1 were used to immunise mice. Theimmunisation procedure is that which is traditionally used: injectioninto mice by intramuscular route (im), of 100 ml of emulsion (1:1) inFreund's complete adjuvant containing 20 mg of product at day 0, with a5 mg Freund's incomplete adjuvant booster on days 21 and 35. The serumfrom the mice is sampled on days −2 and 40 and analysed by ELISA onplates sensitised with the native protein (not chemically treated) andwith kinoid. The sera were tested at a dilution of 1/500.

The results expressed as optical density, obtained from 3 immunised mice(1 to 3) and 3 non-immunised mice (4 to 6), are given in the followingtable:

Native protein Kinoid Mouse 1 D-2 0.1 0.2 D40 1.8 2.1 Mouse 2 D-2 0.150.2 D40 1.5 2.2 Mouse 3 D-2 0.3 0.1 D40 2 2.1 Mouse 4 D-2 0.2 0.15 D400.2 0.1 Mouse 5 D-2 0.15 0.2 D40 0.1 0.15 Mouse 6 D-2 0.15 0.2 D40 0.20.1

These results show that the mice immunised by the kinoid produceantibodies capable of recognising the native protein and the kinoid inthe same fashion. Furthermore, this confirms the non-toxicity ofimmunisation with kinoids as the mice tolerated the immunisation verywell.

EXAMPLE 4 Treatment of TNFα by Carboxamidination

3 mg of TNFα dissolved in 2 ml of 0.3 M Tris buffer, 6 M guanidine, 10mM EDTA, 1 mM DTT were treated with 107 ml of a 0.5 M iodacetamidesolution under a nitrogen barrier for 1 hour and 30 minutes at 37° C.After stopping the reaction by adding 2.5 ml of β-mercaptoethanol, thereaction mixture was dialysed successively against 8 M, 4M, 2 M urea andagainst phosphate buffer (PBS).

EXAMPLE 5 Loss of Biological Activity of the TNFα Prepared byCarboxamidination

There are several tests for measuring the cytotoxic activity of TNFα oncell lines, whether it is the L929 mouse line or the WEHI164 line. Theprinciple of the test on the line WEHI 164 is that the TNFα will inducea cell lysis measured by crystal violet.

50 000 cells are placed in wells of 96-well Costar plates, in 100 ml of10% RPMI FCS. They are left to rest for 4 hours at 37° C. Three times100 ml of RPMI containing increasing dilutions of native or inactivatedTNFα are then added to these wells. The dilutions are carried out 5 by5. After 24 hours at 37° C., the supernatant is removed and the cellsare fixed in each well with 200 ml of methanol for 30 seconds. Afterhaving removed the methanol crystal violet is added in a 1% solutionover 10 minutes. The wells are then washed with distilled water, thenthe plate is allowed to dry by turning it over onto absorbent paper.After drying marking is measured at 620 nm in a plate reader.

The percentage lysis is calculated using the formula:%=(OD _(max) −OD _(exp))/(OD _(max) −OD _(min))×100where max corresponds to the absence of lysis (control RPMI on its own)and min corresponds to maximum lysis (HCl or excess native TNFα).

The results (% lysis) obtained for native TNFα and inactivated TNFαaccording to example 4 are the following:

Quantity/well: Native TNFα Inactivated TNFα 10 mg 100% 5% 1 mg 100% 0%100 ng 100% 5% 10 ng 100% 2% 1 ng 100% 0% 100 pg 98% 3% 10 pg 65% 2% 1pg 5% 4%

It can be seen that, whatever the doses of inactivated TNFα, it nolonger has a lytic effect on the cells (background noise), while thenative TNFα is active up to a dilution up to factor of 10⁵ compared withthe initial dilution.

EXAMPLE 6 Immunogenicity of the Carboxamided TNFα

Kinoids prepared according to example 4 were used to immunise mice. Theimmunisation procedure is that which is traditionally used: injectioninto mice (in im), of 100 ml of emulsion (1:1) in Freund's completeadjuvant containing 20 mg of product at day 0, with a 5 mg Freund'sincomplete adjuvant booster on days 21 and 35. The serum from the miceis sampled on days −2 and 40 and analysed by ELISA on plates sensitisedwith the native protein (not chemically treated) and with kinoid. Thesera were tested at a dilution of 1/500.

The results expressed as optical density, obtained from 3 immunised mice(1 to 3) and 3 non-immunised mice (4 to 6), are given in the followingtable:

Native protein Kinoid Mouse 1 D-2 0.1 0.15 D40 1.9 1.7 Mouse 2 D-2 0.10.2 D40 1.7 2 Mouse 3 D-2 0.2 0.2 D40 1.8 1.8 Mouse 4 D-2 0.2 0.3 D400.1 0.1 Mouse 5 D-2 0.2 0.2 D40 0.15 0.2 Mouse 6 D-2 0.15 0.2 D40 0.20.15

These results show that the mice immunised with the kinoid produceantibodies capable of recognising the native protein and the kinoid inthe same fashion. Furthermore, this confirms the non-toxicity of theimmunisation with the kinoids as the mice tolerated the immunisationvery well.

EXAMPLE 7 Treatment of the IL4 with Elimar's Reagent

1 mg of IL4 in 0.1 M phosphate buffer pH 8.0 was treated with 4 mg ofDTNB for 30 minutes at ambient temperature, away from the light. Themixture was then dialysed against PBS.

EXAMPLE 8 Loss of Biological Activity of the IL4 Treated with Ellman'sReagent

IL4 has several effects on the activation-differentiation ofB-lymphocytes with induction of expression of surface markers such asclass II MHC, CD40, CD23 which can be evalulated by cytofluorometry. Inthe present experiment, the proliferation of mononucleated cells in theperipheral blood (PBMCs) activated by phytohemagglutinin (PHA) and IL2,in the presence of different concentrations of IL4, is calculated.

The PBMCs are purified on a Ficoll gradient. 5 million cells are placedin a flask in 5 ml of RPMI, FCS %, with an addition of PHA 1/1000 andculture is continued over 48 hours at 37° 5% CO₂. IL2 is then added at aconcentration of 20 Ul/ml, and after 48 hours, the cells arecentrifuged, washed and concentrated at 20 000 cells per well in a96-well culture plate in the presence of RPMI, 5% foetal calf serum(FCS), PHA 1/1000. To these wells are added different quantities of IL4diluted with RPMI. The plates are then incubated for 40 hours, thentritiated thymidine is added to them, and after 6 hours, the cellproliferation is measured by evalulating the incorporation of labelledthymidine in a β counter.

The proliferation results (cpm) obtained in the presence of IL4 nativeor inactivated according to example 7 are the following:

Quantity per well: Native IL4 Inactivated IL4 100 ng 49 000 400 10 ng 25000 300 1 ng  6 000 400 0.1 ng  1 000 300

It can be seen that inactivation is effective even after dilution to afactor of 1000, which shows biological inactivation of more than 99.9%.

EXAMPLE 9 Immunogenicity of the IL4 Inactivated by Ellman's Reagent

Kinoids prepared according to example 7 were used to immunise mice. Theimmunisation procedure is that which is traditionally used: injectioninto mice (in im), of 100 ml of emulsion (1:1) in Freund's completeadjuvant containing 20 mg of product at day 0, with a 5 mg Freund'sincomplete adjuvant booster on days 21 and 35. The serum from the miceis sampled on days −2 and 40 and analysed by ELISA on plates sensitisedwith the native protein (not chemically treated) and with kinoid. Thesera were tested at a dilution of 1/500.

The results expressed as optical density, obtained from 3 immunised mice(1 to 3) and 3 non-immunised mice (4 to 6), are given in the followingtable:

native protein kinoid Mouse 1 D-2 0.2 0.1 D40 2 1.9 Mouse 2 D-2 0.3 0.15D40 2.1 1.9 Mouse 3 D-2 0.3 0.2 D40 2.1 1.9 Mouse 4 D-2 0.15 0.2 D40 0.10.25 Mouse 5 D-2 0.1 0.15 D40 0.2 0.2 Mouse 6 D-2 0.1 0.1 D40 0.15 0.3

These results show that the mice immunised with the kinoid produceantibodies capable of recognising the native protein and the kinoid inthe same way. Furthermore, it confirms the non-toxicity of immunisationwith kinoids as the mice tolerated the immunisation very well.

EXAMPLE 10 Treatment of the IL6 by Amidination

40 ml of a 12.5 mg/ml ethylacetimidate solution in 5 N NaOH is added,accompanied by stirring, to 4 ml of a 1 mg/ml IL6 solution in 0.1 Mborate buffer, pH 8.5 cooled in an ice bath. The mixture is stirred for30 to 60 minutes in the ice bath keeping the pH at 8.5 by adding 0.1NNaOH, after which the mixture is dialysed against PBS.

EXAMPLE 11 Loss of Biological Activity of the IL6 Treated by Amidination

The test to measure the activity of IL6 rests on the measurement of theproliferation of a cell line dependant on IL6 for its growth. 7TD1 cellsare cultured in the following culture medium: Dulbecco-modified Eaglemedium (DMEM) with 10% foetal calf serum, 1.5 mM glutamine, 0.24 mMasparagine, 0.55 mM arginine, 50 mM P-mercaptoethanol, 0.1 mMhypoxanthine and 16 mM thymidine. These cells are normally cultured inthe presence of native IL6 at a concentration of 200 U/ml i.e. in theorder of 1 ng/ml of IL6.

The cells are washed twice in DMEM medium alone and resuspended in theculture medium at a concentration of 20 000 cells per ml. 100 ml ofthese cells are added to diluted IL6 (in the same volume of 100 ml) orsample to be tested.

After incubation for 3 to 4 days at 37° C. and 8% CO₂, the number ofliving cells is evaluated by colorimetry, measuring the level ofhexosamimidase (Landegren et al., J immunol Methods, 67, 379, 1984): forthis, the microplates are centrifuged, the cells are washed twice in PBSin order to remove the serum, the colouring substrate is added (1 volumeof 7.5 mM p-nitrophenyl-N-acetyl-b-D-glucosaminide, 0.1 M sodium citratepH 5, 1 volume of 0.5% Triton X100) at 60 ml per well. After incubationfor 4 hours at 37° C., the visualizing agent is added (0.1 Mglycine-NaOH, pH 10.4). The colour is read under 405 nm absorbance witha blank control at 620 nm.

One unit of IL6 corresponds to 50% of the maximum proliferation obtainedaccording to the following calculation:Max prolif 50=control prolif+½ (max prolif-control prolif).

In the case of the preparations of example 10, 5 ng of preparation in100 ml was tested each time, and 3 by 3 dilutions carried out. Thedilutions giving one unit are given in the following table:

Dilution to obtain 1 unit % inactivation Native IL6 3⁶ 0% IL6 kinoid 399.8

These results show the disappearance of IL6 biological activity at morethan 99.8% in the IL6 kinoid inactivated by amidination compared tonative IL6.

EXAMPLE 12 Immunogenicity of the IL6 Inactivated by Amidination

Kinoids prepared according to example 10 were used to immunise mice. Theimmunisation procedure is that which is traditionally used: injectioninto mice (in im), of 100 ml of emulsion (1:1) in Freund's completeadjuvant containing 20 mg of product at day 0, with a 5 mg Freund'sincomplete adjuvant booster on days 21 and 35. The serum from the miceis sampled on days −2 and 40 and analysed by ELISA on plates sensitisedwith the native protein (not chemically treated) and with kinoid. Thesera were tested at a dilution of 1/500.

The results expressed as optical density, obtained from 3 immunised mice(1 to 3) and 3 non-immunised mice (4 to 6), are given in the followingtable:

Native protein Kinoid Mouse 1 D-2 0.15 0.2 D40 1.7 1.9 Mouse 2 D-2 0.10.1 D40 1.9 1.6 Mouse 3 D-2 0.25 0.15 D40 1.9 2 Mouse 4 D-2 0.1 0.1 D400.15 0.1 Mouse 5 D-2 0.2 0.3 D40 0.3 0.2 Mouse 6 D-2 0.2 0.15 D40 0.10.1

These results show that the mice immunised with the kinoid produceantibodies capable of recognising the native protein and the kinoid inthe same fashion. Furthermore, it confirms the non-toxicity of theimmunisation with the kinoids as the mice tolerated the immunisationvery well.

EXAMPLE 13 Treatment of the IL10 by Reductive Alkylation

1.5 mg of sodium borohydride in powder form was added accompanied bystirring to 3 ml of IL10 at a concentration of 1 mg/ml in 0.2 M boratebuffer pH 9. A 37% formaldehyde solution was added to this solution in 5successive portions of 15 ml over a period of 30 minutes. The reactionmedium was dialysed at the end of this period against PBS.

EXAMPLE 14 Loss of Biological Activity of IL10 Inactivated by ReductiveAlkylation

The IL10 has a proliferative action on the cells of the mastocyte mouseline MC/9. MC/9 cells are cultured in RPMI, 10% FCS, 2 mM L-glutamine,0.05 mM β-mercaptoethanol, 100 U/ml of GM-CSF at the initialconcentration of 2 10⁵/ml. After 2 days the cells are washed andresuspended at 2 10⁵/ml in RPMI, 10% FCS.

100 ml of cells are placed in wells of 96-well culture microplates. Tothese wells are added 100 ml of IL10 native or inactivated according toexample 13 at different dilutions. The cells are cultured over 40 hours,then tritiated thymidine is added and after 8 hours the cell DNA iscaught on a filter (MASH, Coulter) and the cell proliferation isevaluated in a β counter.

The proliferation results (cpm) obtained are the following, in relationto the quantity of IL10 poured into each well:

Quantity per well: Native IL10 IL10 kinoid 100 ng 98 000 1 600 10 ng 88000  900 1 ng 36 000 1200 100 pg  6 000 1000 10 pg  1 000  800 control(no IL10) 1100 1100

These results clearly show that the IL10 kinoid was effectivelyinactivated by the reductive alkylation.

EXAMPLE 15 Immunogenicity of the IL10 Inactivated by ReductiveAlkylation

Kinoids prepared according to example 13 were used to immunise mice. Theimmunisation procedure is that which is traditionally used: injectioninto mice (in im), of 100 ml of emulsion (1:1) in Freund's completeadjuvant containing 20 mg of product at day 0, with a 5 mg Freund'sincomplete adjuvant booster on days 21 and 35. The serum from the miceis sampled on days −2 and 40 and analysed by ELISA on plates sensitisedwith the native protein (not chemically treated) and with kinoid. Thesera were tested at a dilution of 1/500.

The results expressed as optical density, obtained from 3 immunised mice(1 to 3) and 3 non-immunised mice (4 to 6), are given in the followingtable:

Native protein Kinoid Mouse 1 D-2 0.2 0.15 D40 2 2.1 Mouse 2 D-2 0.150.3 D40 1.5 1.8 Mouse 3 D-2 0.2 0.1 D40 1.9 1.7 Mouse 4 D-2 0.1 0.1 D400.1 0.15 Mouse 5 D-2 0.15 0.25 D40 0.1 0.1 Mouse 6 D-2 0.2 0.2 D40 0.10.25

These results show that the mice immunised with the kinoid produceantibodies capable of recognising the native protein and the kinoid inthe same manner. Furthermore, this confirms the non-toxicity of theimmunisation with the kinoids as the mice tolerated the immunisationvery well.

EXAMPLE 16 Inactivation of the IFNγ by an Anhydride

A solution of 1M maleic anhydride in redistilled dioxane was added toIFNγ in 0.1 M phosphate buffer, pH 8.1 at a concentration of 1 mg perml, cooled in an ice bath. The addition is carried out, accompanied bystirring, in small fractions at 5 minute intervals, whilst keeping thepH at 8.0 by adding 0.05 N NaOH throughout the reaction time. Thereaction was stopped by dialysis against PBS after stirring wascontinued for a further 30 minutes.

EXAMPLE 17 Immunogenicity of the IFNα Inactivated by an Anhydride

Kinoids prepared according to example 16 were used to immunise mice. Theimmunisation procedure is that which is traditionally used: injectioninto mice (in im), of 100 ml of emulsion (1:1) in Freund's completeadjuvant containing 20 mg of product at day 0, with a 5 mg Freund'sincomplete adjuvant booster on days 21 and 35. The serum from the miceis sampled on days −2 and 40 and analysed by ELISA on plates sensitisedwith the native protein (not chemically treated) and with kinoid. Thesera were tested at a dilution of 1/500.

The results expressed as optical density, obtained from 3 immunised mice(1 to 3) and 3 non-immunised mice (4 to 6), are given in the followingtable:

Native protein Kinoid Mouse 1 D-2 0.3 0.2 D40 2.2 1.9 Mouse 2 D-2 0.20.1 D40 1.9 1.7 Mouse 3 D-2 0.15 0.25 D40 1.9 2.1 Mouse 4 D-2 0.15 0.1D40 0.1 0.25 Mouse 5 D-2 0.25 0.1 D40 0.15 0.15 Mouse 6 D-2 0.1 0.2 D400.15 0.1

These results show that the mice immunised with the kinoid produceantibodies capable of recognising the native protein and the kinoid inthe same fashion. Furthermore, it confirms the non-toxicity ofimmunisation with kinoids as the mice tolerated the immunisation verywell.

EXAMPLE 18 Treatment of the IL13 with p-hydroxyphenylglyoxal

100 ml of a 20 mg/ml solution of hydroxyphenylglyoxal was added to a 1mg/ml solution of IL13 in PBS, in the same buffer per ml of IFNγsolution. The reaction was continued for 1 hour at a temperature of 37°C., and the reaction medium was dialysed against PBS.

EXAMPLE 19 Loss of Biological Activity of the IL13 Treated withp-hydroxyphenylglyoxal

The IL13 has a proliferative activity on the erythroleucemic human cellline TF-1. As in the previous experiments, the TF-1 cells were culturedin RPMI, 5% FCS, then distributed onto microplates at 10⁴ cells per wellin 100 ml. Increasing dilutions of IL13, native or inactivated accordingto example 18, are added to these wells. After 40 hours, tritiatedthymidine is added.

After 8 hours, the DNA from the cells is collected on a filter (MASH,Coulter) and then the radioactivity is measured with a β counter.

The proliferation results (in cpm) obtained with the IL13 are thefollowing:

Quantity added per well native IL13 IL13 kinoid 100 ng 76 000 2000 10 ng80 000 1 200 1 ng 62 000 900 100 pg 21 000 700 10 pg 3 000 1 100 control900 900

It can be seen that the inactivation was effective since the native IL13diluted 10⁴ times (10 pg) is more active than the non-diluted kinoid atthe same initial concentration (100 ng).

EXAMPLE 20 Immunogenicity of the IL13 Inactivated byp-hydroxyphenylglyoxal

Kinoids prepared according to example 18 were used to immunise mice. Theimmunisation procedure is that which is traditionally used: injectioninto mice (in im), of 100 ml of emulsion (1:1) in Freund's completeadjuvant containing 20 mg of product at day 0, with a 5 mg Freund'sincomplete adjuvant booster on days 21 and 35. The serum from the miceis sampled on days −2 and 40 and analysed by ELISA on plates sensitisedwith the native protein (not chemically treated) and with kinoid. Thesera were tested at a dilution of 1/500.

The results expressed as optical density, obtained from 3 immunised mice(1 to 3) and 3 non-immunised mice (4 to 6), are given in the followingtable:

Native protein Kinoid Mouse 1 D-2 0.3 0.25 D40 1.8 1.8 Mouse 2 D-2 0.20.15 D40 2.1 2 Mouse 3 D-2 0.35 0.15 D40 2 1.8 Mouse 4 D-2 0.3 0.1 D400.1 0.1 Mouse 5 D-2 0.1 0.25 D40 0.2 0.15 Mouse 6 D-2 0.2 0.1 D40 0.30.15

These results show that the mice immunised with the kinoid produceantibodies capable of recognising the native protein and the kinoid inthe same fashion. Furthermore, it confirms the non-toxicity of theimmunisation with the kinoids as the mice tolerated the immunisationvery well.

EXAMPLE 21 Inactivation of the IL4 by Reductive Alkylation

1 mg of sodium borohydride and, immediately afterwards 50 ml of 37%formaldehyde solution were added accompanied by stirring in 5 successivefractions at 5 minute intervals to a solution of IL4 at a concentrationof 1 mg/ml (5 ml) in 0.2 M borate buffer at pH 9. After the lastaddition of the aldehyde, the mixture was stirred for a further 15minutes before being dialysed against PBS.

The same test was carried out as that described in example 8 to measurethe inactivation of the IL4 kinoid by reductive alkylation. The resultobtained was similar to that of example 8 as the cells stimulated bynative IL4 proliferated and not the cells stimulated by the kinoid.

EXAMPLE 22 Immunogenicity of the IL4 Inactivated by Reductive Alkylation

Kinoids prepared according to example 21 were used to immunise mice. Theimmunisation procedure is that which is traditionally used: injectioninto mice (in im), of 100 ml of emulsion (1:1) in Freund's completeadjuvant containing 20 mg of product at day 0, with a 5 mg Freund'sincomplete adjuvant booster on days 21 and 35. The serum from the miceis sampled on days −2 and 40 and analysed by ELISA on plates sensitisedwith the native protein (not chemically treated) and with kinoid. Thesera were tested at a dilution of 1/500.

The results expressed as optical density, obtained from 3 immunised mice(1 to 3) and 3 non-immunised mice (4 to 6), are given in the followingtable:

Native protein Kinoid Mouse 1 D-2 0.25 0.2 D40 1.7 2 Mouse 2 D-2 0.10.15 D40 1.8 1.8 Mouse 3 D-2 0.1 0.2 D40 2 1.9 Mouse 4 D-2 0.25 0.1 D400.15 0.2 Mouse 5 D-2 0.2 0.2 D40 0.3 0.1 Mouse 6 D-2 0.1 0.1 D40 0.150.1

These results show that the mice immunised with the kinoid produceantibodies capable of recognising the native protein and the kinoid inthe same fashion. Furthermore, it confirms the non-toxicity ofimmunisation with kinoids as the mice tolerated the immunisation verywell.

EXAMPLE 23 Inactivation of the TNFα with Ethylacetimidate.

50 ml of an ethylacetimidate solution, and HCl at 12.5 mg/ml in 5N NaOHcooled in an ice bath were added, accompanied by stirring to 2 ml of a 1mg/ml solution of IFNα in 0.1 M borate buffer. The reaction wascontinued for 1 hour at 0° C., accompanied by stirring, keeping the pHat 8.5 by adding diluted NaOH. The reaction was stopped by dialysisagainst PBS.

The inactivation of the TNFα obtained was evaluated according to thesame test as that described in example 5. The results were similar andthere was no longer lysis of the cells in the presence of the kinoid.

EXAMPLE 24 Immunogenicity of the TNFα Inactivated with Ethylacetimidate

Kinoids prepared according to example 23 were used to immunise mice. Theimmunisation procedure is that which is traditionally used: injectioninto mice (in im), of 100 ml of emulsion (1:1) in Freund's completeadjuvant containing 20 mg of product at day 0, with a 5 mg Freund'sincomplete adjuvant booster on days 21 and 35. The serum from the miceis sampled on days −2 and 40 and analysed by ELISA on plates sensitisedwith the native protein (not chemically treated) and with kinoid. Thesera were tested at a dilution of 1/500.

The results expressed as optical density, obtained from 3 immunised mice(1 to 3) and 3 non-immunised mice (4 to 6), are given in the followingtable:

Native protein Kinoid Mouse 1 D-2 0.1 0.2 D40 2.1 2 Mouse 2 D-2 0.1 0.2D40 2 2.2 Mouse 3 D-2 0.2 0.15 D40 2.1 1.9 Mouse 4 D-2 0.25 0.1 D40 0.20.2 Mouse 5 D-2 0.2 0.15 D40 0.15 0.2 Mouse 6 D-2 0.3 0.1 D40 0.1 0.25

These results show that the mice immunised with the kinoid produceantibodies capable of recognising the native protein and the kinoid inthe same fashion. Furthermore, it confirms the non-toxicity of theimmunisation with the kinoids as the mice tolerated the immunisationvery well.

EXAMPLE 25 Treatment of the IL1 by Carboxymethylation

2 mg of IL1 was dissolved in 2 ml of 0.3 M Tris buffer, pH 8 containing6 M guanidine and 10 mM dithiothreitol (DTT) previously deaerated bypurging with nitrogen. 40 ml of a deaerated solution of 0.5 M iodaceticacid was added to this solution and the reaction mixture was incubatedfor 90 minutes at 37° C. under a nitrogen barrier. The reaction was thenstopped by adding 10 ml of a 1:10 solution of P-mercaptoethanol.Incubation was extended for another hour and the reaction mixture wasdialysed successively against 8 M, 4 M, 2 M urea and finally againstPBS.

EXAMPLE 26 Loss of Biological Activity of the IL1 Treated byCarboxymethylation

The biological activity of IL1 (IL1 a or IL1) can be shown using cellsfrom the NOB mouse line which are induced to produce IL2 in the presenceof IL1. The cells are cultured in RPMI, 5% FCS. In the growth phase,they are washed (centrifugation-washing) and resuspended at aconcentration of 10⁵ cells/ml. The cells are then aliquoted onmicroplates at 100 ml per well, 100 ml of IL1, native or treatedaccording to example 25 is added to them in increasing dilutions. Theplates are then incubated at 37° C. for 36 hours and the production ofIL2 in the supernatant is measured by ELISA (Qantikine, R&DDiagnostics).

The results obtained (in optical density) for the IL1 and the kinoid ofexample 25 are the following:

Quantity per well: Native IL1 Kinoid 1 mg 2.1 0.4 100 pg 2.2 0.2 10 pg1.3 0.3 1 pg 0.6 0.2 0.1 pg 0.3 0.2 control 0.2 0.2

It is thus seen that the kinoid has lost its activity compared with thenative IL1 by more than a factor of 1000.

EXAMPLE 27 Immunogenicity of the Carboxymethylated IL1

Kinoids prepared according to example 25 were used to immunise mice. Theimmunisation procedure is that which is traditionally used: injectioninto mice (in im), of 100 ml of emulsion (1:1) in Freund's completeadjuvant containing 20 mg of product at day 0, with a 5 mg Freund'sincomplete adjuvant booster on days 21 and 35. The serum from the miceis sampled on days −2 and 40 and analysed by ELISA on plates sensitisedwith the native protein (not chemically treated) and with kinoid. Thesera were tested at a dilution of 1/500.

The results expressed as optical density, obtained from 3 immunised mice(1 to 3) and 3 non-immunised mice (4 to 6), are given in the followingtable:

Native protein Kinoid Mouse 1 D-2 0.3 0.2 D40 1.6 1.7 Mouse 2 D-2 0.20.3 D40 1.8 2 Mouse 3 D-2 0.1 0.2 D40 1.9 1.6 Mouse 4 D-2 0.15 0.2 D400.1 0.25 Mouse 5 D-2 0.1 0.2 D40 0.3 0.3 Mouse 6 D-2 0.25 0.15 D40 0.30.1

These results show that the mice immunised with the kinoid produceantibodies capable of recognising the native protein and the kinoid inthe same fashion. Furthermore, it confirms the non-toxicity ofimmunisation with kinoids as the mice tolerated the immunisation verywell.

EXAMPLE 28 Inactivation of the IFNα by Maleimidation

50 of a 2 mg/ml solution of β-maleimidopropionic acid is addedaccompanied by stirring the same buffer, to 1.7 ml of a 1 mg/ml solutionof IFNα in 30 mM buffer, 1 mM EDTA, pH 7.8. After reaction, for 15minutes at laboratory temperature, the reaction mixture was dialysedagainst PBS.

The degree of inactivation of the maleimidated IFNα was evaluated by thetest of inhibition of the lysis of the MDBK cells with the VSV virus(the same test as for IFNγ in example 2). The biological activity of thekinoid was inhibited by more than 99.9% compared with native IFNα.

EXAMPLE 29 Immunogenicity of the Maleimidated IFNα

Kinoids prepared according to example 28 were used to immunise mice. Theimmunisation procedure is that which is traditionally used: injectioninto mice (in im), of 100 ml of emulsion (1:1) in Freund's completeadjuvant containing 20 mg of product at day 0, with a 5 mg Freund'sincomplete adjuvant booster on days 21 and 35. The serum from the miceis sampled on days −2 and 40 and analysed by ELISA on plates sensitisedwith the native protein (not chemically treated) and with kinoid. Thesera were tested at a dilution of 1/500.

The results expressed as optical density, obtained from 3 immunised mice(1 to 3) and 3 non-immunised mice (4 to 6), are given in the followingtable:

Native protein Kinoid Mouse 1 D-2 0.1 0.25 D40 1.7 2 Mouse 2 D-2 0.150.15 D40 1.9 2.1 Mouse 3 D-2 0.35 0.3 D40 1.8 1.9 Mouse 4 D-2 0.15 0.1D40 0.25 0.3 Mouse 5 D-2 0.1 0.3 D40 0.2 0.15 Mouse 6 D-2 0.25 0.1 D400.2 0.1

These results show that the mice immunised with the kinoid produceantibodies capable of recognising the native protein and the kinoid inthe same fashion. Furthermore, it confirms the non-toxicity ofimmunisation with kinoids as the mice tolerated the immunisation verywell.

1. A chemically modified TNF-α cytokine of formulaCy—(X—R)_(n) in which Cy represents native TNF-α cytokine having toxicbiological properties —(XR) represents a chemically-reactive function ofan amino acid contained in said native TNF-α cytokine or where Xrepresents an NH group or a sulphur atom, n represents an integer from 1to 70, and R is either (i) a carboxymethylated, carboxamide,N-alkylmaleimide, thionitrobenzoic group or a radical formed by reactionof a thiol function with ammonium 4-chloro-7-sulphobenzofurazane,N-[iodoethyl]-trifluroacetamide orN-(6-[7-amino-4-methylcoumarin-3-acetamido]hexyl)-3′-(2′-pyridyldithio)propionamide; or (ii) a radical formed by reaction of an amino functionwith ethylacetimidate, an anhydride, 2-iminothiolane-HCl, N-succinimidylS-acetylthioacetate, sulphosuccinimidyl-acetate,sulphosuccinimidyl-4-O-[4-4′-dimethoxytrityl]butyrate, succinimidyl7-amino-4-methlcoumarin-3-acetate, sulphosuccinimidyl7-amino-4-methylcoumarin-3-acetate or phenylglyoxal, said chemicallymodified TNF-α cytokine elicits antibodies which neutralize or blocksaid native TNF-α cytokine and at the same time bringing about the lossof at least 90% of the toxic biological properties of said native TNF-αcytokine.
 2. A chemically modified TNF-α cytokine according to claim 1,wherein at least 70% of the X groups are modified in the form of an—(XR) group.
 3. A pharmaceutical composition, comprising as activeingredient the chemically modified TNF-α cytokine of claim 1 and apharmaceutically acceptable excipient.
 4. A pharmaceutical composition,comprising the chemically modified TNF-α cytokine of claim 1 in atherapeutic amount and a pharmaceutically acceptable excipient.
 5. Avaccine, comprising the chemically modified TNF-α cytokine of claim 1.6. The chemically modified TNF-α of claim 1 which is carboxyamidatedTNF-α.
 7. The chemically modified TNF-α cytokine of claim 1 which isethylacetimidated TNF-α.